aSyn: a prion-like protein?

Recently, the possibility that the progression of aSyn pathology in synucleinopathies would be mediated through cell-to-cell spreading of oligomers or aggregates has become an attractive model to explain how aSyn related toxicity and neurodegeneration might propagate throughout neuroanatomically connected regions of the diseased brain.

Braak and colleagues hypothesized about the progressive, stereotypic spread of Lewy pathology by histopathological studies of PD post-mortem brains¹¹. In the early stage of the disease, aSyn positive LB and LNs accumulate mainly in the olfactory bulb and in the dorsal motor nucleus of the vagus and then they spread in anatomically connected regions with the progression of the disease^50,122 (Figure 7).

Six neuropathological stages of PD were described, with increasing number of regions that successively exhibit aSyn pathology, proposing a model where a “spreading agent” (e.g. a neurotrophic virus) might propagate through a defined neuronal pathway, in a prion-like manner¹²³.

A supportive observation to this theory resulted from an experimental treatment in which embryonic dopaminergic neurons were transplanted into the putamen of human PD patients^124,125. The finding of LB-like inclusions in these exogenously grafted neuronal population significantly influenced the field, leading to the formulation of the prion-like hypothesis of aSyn, which postulated that misfolded aSyn is transferred between connected cells, acting as a template to initiate aggregation of endogenous protein in recipient and otherwise healthy neurons^50,126,127.

Figure 7. Braak´s staging hypothesis for the progression of PD pathology.

A key premise for the prion-like hypothesis is that aSyn assemblies can be taken up by neurons, transported along the axons and finally transferred to neighbouring cells through different ways, (i.e. being released into the extracellular space)¹²⁸.

So far, numerous in vitro and in vivo studies have addressed different aspects of this complex series of events^80,128–136.

First, it has been widely demonstrated that exogenously added aSyn oligomers and aggregates can bind to the surface of cultured cells, both in their free state or associated in extracellular vesicles, due to the interaction with membranous proteins^137,138, or with lipid components of the membranes¹³⁹ (i.e., proteoglycans).

Several mechanisms for the internalization of aSyn have been proposed, some of them appearing to be assembly-state specific. Monomeric aSyn seems to be able to penetrate cell membranes and passively diffuse into cells¹²⁹, while larger assemblies utilize specific pathways, including receptor-mediated endocytosis¹³⁹.

It has been shown that exogenous aSyn further induced neuronal cell death through Rab 5A-dependent endocytosis¹⁴⁰, while another study reported that heparan-sulphate proteoglycans mediate macropinocytosis of aSyn and other aggregation prone proteins¹³⁹.

Several findings showed that aSyn oligomers and fibrillary species can enter cells via dynamin-dependent endocytosis, and that absorptive endocytosis (an intermediate process between fluid-phase endocytosis and receptor-mediated endocytosis) promoted the uptake of both tau and aSyn fibrils^50,134,141. However, due to the size of fibrillar aggregates, seems unlikely to consider receptor-mediated endocytosis - which requires the interaction between ligands and cell-surface receptors - as a major mode of fibril internalization¹⁴².

Based on the above-mentioned mechanisms, it looks like a direct cell contact is not necessarily required for the propagation of aSyn aggregates. Nonetheless, recent evidence suggests that tunnelling nanotubes could also be involved in the spreading of aSyn¹⁴³. These structures would provide a useful channel for the migration from one cell to another, obviating the need for those misfolded proteins, otherwise enclosed within an endosome, to cross plasma membrane and to gain access to the cytosolic compartment (Figure 8).

Once inside the naïve cells, exogenous, misfolded aSyn assemblies can amplify by recruiting and triggering the aggregation of endogenous, cytosolic aSyn¹²⁸. Many efforts in the last decade were aimed at shedding light into the process responsible of the structural conversion of aSyn, suggesting that endogenous aSyn aggregates through a seeding process where the imported aSyn act as a template. The intrinsic

structure of the seed is preserved by structurally well-defined longitudinal and lateral molecular interaction between newly recruited aSyn monomers and the terminal, exposed part of the seed, through the use of a “lock and dock” mechanism, as described above.

Part of the aSyn assemblies can also be degraded by lysosomes¹²⁹, which is particularly interesting considering that dysfunction and impairment of the autophagy-lysosomal pathway (ALP) are strictly linked to PD pathogenesis.

Transmission of aSyn fibrils from neuron to neuron can undergo anterograde and retrograde transport, and some of the monitored movement of aSyn assemblies occurs at a velocity consistent with fast axonal transportation¹³⁵.

Figure 8. Potential proposed mechanisms mediating cell-to-cell transmission of aSyn.

Misfolded protein seeds in the form of oligomers or protofibrils are initially formed in the cytoplasm of the donor neuron, where soluble monomers are recruited into larger aggregates. A large number of propagating seeds can be generated through fragmentation of the existing fibrillary species, or through secondary nucleation. Protein aggregates can be released in the extracellular space in a “naked” form (a) or via exosomes (b). aSyn can passively diffuse trough the plasma membrane or (1) enter the recipient cell by fluid-phase endocytosis (2) or receptor-mediated endocytosis, (3) as well as through the fusion of exosomal vesicles with the recipient cell. (4) The transfer of protein could also happen via nanotubes, structures that connect directly the cytoplasm of two cells (5). Internalized, misfolded protein then recruit

Regarding the release of aSyn, intracellular aSyn aggregates can be secreted into the extracellular space and therefore uptaken by neighbouring neurons, microglia or astrocytes133,135,136.

The first report highlighting the detection of aSyn in the extracellular space even preceded the prion-like propagation hypothesis, and was based on the observation of aSyn molecules in biological fluids - including cerebrospinal fluid (CSF) and blood plasma - of both healthy and PD subjects¹⁴⁴. These results imply that aSyn is typically available in the extracellular space in the CNS, regardless of a pathological state.

Further results obtained from cultured cells confirmed that aSyn can be secreted in an active way and that the secretion can be constitutive or regulated⁵⁰. Furthermore, higher cytoplasmic level of the protein - as well as inhibition of the proteasomal and lysosomal system¹²⁸ - lead to an increased release of aSyn, probably due to some compensative mechanism necessary to keep balanced the cytosolic level of the protein.

Pathways leading to the release of toxic aSyn oligomers include exocytosis in clear vesicles¹²⁹, exosomal release^145,146 and penetration from the donor cell membrane¹²¹. Another way through which aSyn can reach the extracellular moiety is throughout necrotic cell death, although there are still very little evidences suggesting that the amount of aSyn released from these cells and its contribution to the extracellular, pathological pool would be determinant for the propagation of the disease⁵⁰.

Overall, cellular stress, proteasomal and mitochondrial dysfunction¹³¹ as well as overexpression and cytosolic accumulation seems to drive aSyn secretion from neural cells, supporting the general idea that aSyn exocytosis may increase in the affected brains, bringing a fundamental contribution to the disease progression.

Another essential process for the prion-like hypothesis is the propagation of misfolded aSyn and the spreading of the related neuropathology in different, distant brain region.

This was shown in multiple experiments in rodent and nonhuman primate, using intracerebral injection of brain homogenates from PD and DLB patients, brain tissues from transgenic animal with aSyn pathology, and preparation of pre-formed fibrils (PFFs) generated from recombinant aSyn141,147–149.

Injection of aSyn PFFs into the striatum not only lead to aggregation of endogenous aSyn, but it also had drastic consequences on the viability of dopaminergic neurons, leading to a widespread synaptic dysfunction¹³⁴. Of note, few months after the injection aSyn aggregates had spread throughout the brain, while mice injected with vehicle remained healthy and free of pathology, indicating that the presence of misfolded, exogenously added aSyn is sufficient to trigger and spread aSyn pathology¹⁵⁰.

One of the advantages of using recombinant protein is that the seeding process can be controlled carefully in the test tube before the intracellular injection is made, which does not apply to a crude mix such a brain homogenate.

Indeed, one of the latest debate in the field is related to the existence of different

“strains” of aSyn aggregates and whether their presence could explain the different morphological conformation - as well as the cellular and anatomical predilection - of aSyn inclusions in different synucleinopathies¹⁵¹.

Intracerebral or systemic injections of fractionated brain homogenates from PD or MSA patients in animal models induce distinct neuropathology with “strain-specific”

features and characteristic that resemble the pathology of origin, adding further support to the idea that different types of aSyn fibrillary assemblies exhibit different toxicity and give rise to different type of neuropathology.